Effect of mutating subsite -7 on product specificity of cyclodextrin glucanotransferase from alkalophilic Bacillus clarkii.
- Author:
Dong YANG
1
;
Jingfei TIAN
;
Sheng CHEN
;
Jing WU
Author Information
1. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
- Publication Type:Journal Article
- MeSH:
Bacillus;
enzymology;
genetics;
Escherichia coli;
genetics;
metabolism;
Glucosyltransferases;
biosynthesis;
genetics;
Mutant Proteins;
biosynthesis;
genetics;
Recombinant Proteins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2012;28(2):191-202
- CountryChina
- Language:Chinese
-
Abstract:
To investigate the mechanism of high product specificity of gamma-clodextrin glucanotransferase (CGTase) from alkalophilic Bacillus clarkii 7364, we aligned protein sequence and structure model, found out that loss of 6 amino acids at subsite -7 probably affected its product specificity. Using overlapping PCR method, we inserted 6 amino acids into subsite -7 of CGTase. The mutant CGTase gene was ligated with pET-20b (+) and expressed in Escherichia coli BL21 (DE3). The extracellular recombinant enzyme was used to transform soluble starch into cyclodextrins (CDs). HPLC analysis results show that, compared to wild CGTase, the gamma-CDs produced by mutant enzyme decreased from 76.0% to 12.5%, whereas the ratio of alpha- and beta-CDs increased from 8.7% and 15.2% to 37.5% and 50%. The possible mechanism was that, compared to alpha-, beta-CGTase, wild gamma-CGTase lacks 6 amino acids in its subsite -7. This conformation provided more space for glucose combination and was thus advantageous for forming gamma-CD. When the 6 amino acids were inserted into the subsite -7 of wild gamma-CGTase, the space to bind with glucose reduced and consequently resulted in less gamma-CD production.
